Ken-ichi Hirata

Overexpression of endothelial nitric oxide synthase accelerates atherosclerotic lesion formation in apoE-deficient mice

Nitric oxide (NO) derived from endothelial NO synthase (eNOS) is regarded as a protective factor against atherosclerosis. Therefore, augmentation of eNOS expression or NO production by pharmacological intervention is postulated to inhibit atherosclerosis. We crossed eNOS-overexpressing (eNOS-Tg) mice with atherogenic apoE-deficient (apoE-KO) mice to determine whether eNOS overexpression in the endothelium could inhibit the development of atherosclerosis. After 8 weeks on a high-cholesterol diet, the atherosclerotic lesion areas in the aortic sinus were unexpectedly increased by more than twofold in apoE-KO/eNOS-Tg mice compared with apoE-KO mice. Also, aortic tree lesion areas were approximately 50% larger in apoE-KO/eNOS-Tg mice after 12 weeks on a high-cholesterol diet. Expression of eNOS and NO production in aortas from apoE-KO/eNOS-Tg mice were significantly higher than those in apoE-KO mice. However, eNOS dysfunction, demonstrated by lower NO production relative to eNOS expression and enhanced superoxide production in the endothelium, was observed in apoE-KO/eNOS-Tg mice. Supplementation with tetrahydrobiopterin, an NOS cofactor, reduced the atherosclerotic lesion size in apoE-KO/eNOS-Tg mice to the level comparable to apoE-KO mice, possibly through the improvement of eNOS dysfunction. These data demonstrate that chronic overexpression of eNOS does not inhibit, but accelerates, atherosclerosis under hypercholesterolemia and that eNOS dysfunction appears to play important roles in the progression of atherosclerosis in apoE-KO/eNOS-Tg mice.

High density lipoprotein reverses inhibitory effect of oxidized low density lipoprotein on endothelium-dependent arterial relaxation.

We have recently reported that oxidized low density lipoprotein (ox-LDL) inhibits endothelium-dependent arterial relaxation through its increased lysophosphatidylcholine (LPC). In this study we examined whether high density lipoprotein (HDL) has any effect on the inhibition of endothelium-dependent relaxation by ox-LDL in isolated strips of rabbit thoracic aorta. Both low density lipoprotein (LDL) and HDL were isolated from normal human plasma, and LDL was oxidized by exposure to copper. Preincubation of arterial strips with ox-LDL (0.1-0.5 mg protein/ml) inhibited endothelium-dependent relaxation to acetylcholine (ACh) in a concentration-dependent manner. HDL (1 mg protein/ml) by itself had no effect on the relaxation to ACh. In the presence of HDL, the inhibition by ox-LDL was markedly reduced. Although synthetic L-alpha-palmitoyl LPC (5 micrograms/ml) completely abolished a relaxation to ACh, the preincubation of arterial strips with HDL completely prevented the LPC-induced inhibition. Moreover, a relaxation to ACh was almost completely recovered when the strips were washed with buffer containing HDL even after LPC-induced inhibition had occurred. HDL markedly reduced the incorporation of [1-14C]palmitate-labeled LPC ([14C]LPC) into cultured bovine aortic endothelial cells and promoted the release of cell-incorporated [14C]LPC into the medium, resulting in a reduction of the remaining [14C]LPC in the cells. Agarose electrophoresis after incubation of a mixture of ox-LDL labeled with [14C]LPC and unlabeled HDL demonstrated a transfer of [14C]LPC from ox-LDL to HDL. These results indicate that HDL reverses the ox-LDL-induced impairment of endothelium-dependent relaxation by removing LPC from ox-LDL and preventing LPC from acting on the endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced Hypoxic Pulmonary Vascular Remodeling by Nitric Oxide From the Endothelium

We examined whether overproduction of endogenous nitric oxide (NO) can prevent hypoxia-induced pulmonary hypertension and vascular remodeling by using endothelial NO-overexpressing (eNOS-Tg) mice. Male eNOS-Tg mice and their littermates (wild-type, WT) were maintained in normoxic or 10% hypoxic condition for 3 weeks. In normoxia, eNOS protein levels, Ca2+-dependent NOS activity, and cGMP levels in the lung of eNOS-Tg mice were higher than those of WT mice. Activity of eNOS and cGMP production in the lung did not change significantly by hypoxic exposure in either genotype. Chronic hypoxia did not induce iNOS expression nor increase its activity in either genotype. Plasma and lung endothelin-1 levels were increased by chronic hypoxia, but these levels were not significantly different between the 2 genotypes. In hemodynamic analysis, right ventricular systolic pressure (RVSP) in eNOS-Tg mice was similar to that in WT mice in normoxia. Chronic hypoxia increased RVSP and induced right ventricular hypertrophy in both genotypes; however, the degrees of these increases were significantly smaller in eNOS-Tg mice. Histological examination revealed that hypoxic mice showed medial wall thickening in pulmonary arteries. However, the increase of the wall thickening in small arteries (diameter <80 &mgr;m) by chronic hypoxia was inhibited in eNOS-Tg mice. Furthermore, muscularization of small arterioles was significantly attenuated in eNOS-Tg mice. Thus, we demonstrated directly that overproduction of eNOS-derived NO can inhibit not only the increase in RVSP associated with pulmonary hypertension but also remodeling of the pulmonary vasculature and right ventricular hypertrophy induced by chronic hypoxia.

Lysophosphatidylcholine inhibits bradykinin-induced phosphoinositide hydrolysis and calcium transients in cultured bovine aortic endothelial cells.

Vascular endothelium, which produces endothelium-derived relaxing and constricting factors, plays an important role in regulating the vascular tone. We recently demonstrated that oxidized low density lipoprotein inhibited endothelium-dependent relaxation and that lysophosphatidylcholine accumulated during the oxidative modification of low density lipoprotein was the essential substance for the inhibition of endothelium-dependent relaxation. To clarify the mechanisms of the inhibitory effect of lysophosphatidylcholine, we used a bioassay system to investigate the effect of lysophosphatidylcholine on the production and/or release of endothelium-derived relaxing factor and its effect on the cytosolic Ca2+ level ([Ca2+]i) and phosphoinositide hydrolysis in cultured bovine aortic endothelial cells. [Ca2+]i was monitored by the fura 2 method, and the accumulation of inositol phosphates in cells labeled with myo-[2-3H]inositol was measured. Bioassay experiments showed that lysophosphatidylcholine inhibited the production and/or release of endothelium-derived relaxing factor from cultured endothelial cells. Lysophosphatidylcholine (5-20 micrograms/ml) induced a biphasic increase in [Ca2+]i, which consisted of a rapid increase followed by a sustained increase, and the initial component was a result of mobilization from intracellular Ca2+ stores without detectable synthesis of inositol 1,4,5-trisphosphates. Furthermore, lysophosphatidylcholine (5-20 micrograms/ml) dose-dependently inhibited both phosphoinositide hydrolysis and the increases in [Ca2+]i evoked by bradykinin. These results indicate that the impairment of endothelium-dependent relaxation induced by lysophosphatidylcholine is due to the inhibition of phosphoinositide hydrolysis and the subsequent increases in [Ca2+]i in endothelial cells. Lysophosphatidylcholine that accumulates in oxidized low density lipoprotein and atherosclerotic arteries may play an important role in the modification of endothelial function.

Mechanisms of Reduced Nitric Oxide/cGMP–Mediated Vasorelaxation in Transgenic Mice Overexpressing Endothelial Nitric Oxide Synthase

NO, constitutively produced by endothelial NO synthase (eNOS), plays a key regulatory role in vascular wall homeostasis. We generated transgenic (Tg) mice overexpressing eNOS in the endothelium and reported the presence of reduced NO-elicited relaxation. The purpose of this study was to clarify mechanisms of the reduced response to NO-mediated vasodilators in eNOS-Tg mice. Thoracic aortas of Tg and control mice were surgically isolated for vasomotor studies. Relaxations to acetylcholine and sodium nitroprusside were significantly reduced in Tg vessels compared with control vessels. Relaxations to atrial natriuretic peptide and 8-bromo-cGMP were also significantly reduced in Tg vessels. Reduced relaxations to these agents were restored by chronic N(G)-nitro-L-arginine methyl ester treatment. Basal cGMP levels of aortas were higher in Tg mice than in control mice, whereas soluble guanylate cyclase (sGC) activity in Tg vessels was approximately 50% of the activity in control vessels. Moreover, cGMP-dependent protein kinase (PKG) protein levels and PKG enzyme activity were decreased in Tg vessels. These observations indicate that chronic overexpression of eNOS in the endothelium resulted in resistance to the NO/cGMP-mediated vasodilators and that at least 2 distinct mechanisms might be involved: one is reduced sGC activity, and the other is a decrease in PKG protein levels. We reported for the first time that increased NO release from the endothelium reduces sGC and PKG activity in mice. These data may provide a new insight into the mechanisms of nitrate tolerance and cross tolerance to nitrovasodilators.

Endothelial NO Synthase Overexpression Inhibits Lesion Formation in Mouse Model of Vascular Remodeling

Abstract —NO produced by endothelial NO synthase (eNOS) plays important roles in the regulation of vascular tone and structure. The purpose of this study was to clarify the role of eNOS-derived NO on vascular remodeling by use of eNOS-transgenic (eNOS-Tg) mice. The common carotid artery was ligated just proximal to the carotid bifurcation. Four weeks later, the proximal carotid artery of the ligation site was histologically examined. In this vascular remodeling model, the endothelium remains uninjured, but neointimal and medial thickening occurs in combination with a reduction in vascular diameter at the proximal portion of the ligation. At 4 weeks after ligation, the respective neointimal and medial areas in wild-type mice were 17 200±1100 and 24 300±1500 &mgr;m2, whereas both were reduced to 8000±1900 (P <0.01) and 18 400±700 &mgr;m2 (P <0.01) in eNOS-Tg mice (n=8). Total vascular area was not different between the 2 genotypes. NG-Nitro-l-arginine methyl ester treatment increased neointimal and medial areas to the same extent in both genotypes. Leukocyte infiltration was observed in the luminal side of the vessel, but the number of infiltrating cells was significantly attenuated in eNOS-Tg mice compared with wild-type mice. This reduction of leukocyte infiltration in eNOS-Tg mice was associated with reduced expressions of intracellular adhesion molecule-1 and vascular cellular adhesion molecule-1 on the endothelium. In conclusion, chronic eNOS overexpression in the endothelium reduced leukocyte infiltration and inhibited neointimal formation and medial thickening. Our data provide the evidence for the regulatory role of NO from the endothelium on vascular structure integrity.

Expression of G2A, a Receptor for Lysophosphatidylcholine, by Macrophages in Murine, Rabbit, and Human Atherosclerotic Plaques

Objective—Lysophosphatidylcholine (LPC), a major phospholipid component of oxidized low density lipoprotein, has been demonstrated to induce multiple functional alterations of vasculature that are potentially involved in atherosclerosis. Recently, an orphan G-protein-coupled receptor, G2A, has been identified as a high-affinity receptor for LPC. Although it has been demonstrated that G2A is expressed predominantly in lymphoid tissues and lymphocytes, there are no reports to determine whether G2A is expressed in atherosclerotic lesions and cardiovascular cells. Methods and Results—Immunohistochemistry with an anti-G2A antibody revealed that G2A was expressed predominantly by macrophages within atherosclerotic lesions at the aortic root of apolipoprotein E-deficient mice and the thoracic aortas of Watanabe heritable hyperlipidemic rabbits. In atherosclerotic plaques of human coronary arterial specimens, G2A was expressed by macrophages within the lipid-rich plaques, whereas no immunoreactivity of G2A was observed in fibrous plaques where macrophages did not exist. Reverse transcription-polymerase chain reaction analysis demonstrated that G2A mRNA was highly expressed in human and murine monocytes/macrophages. The expression of G2A protein was detected in human and murine monocytes/macrophages by immunoblotting. Conclusions—These findings demonstrate that monocytes/macrophages abundantly express G2A and suggest that G2A may play a role in the formation and progression of atherosclerotic lesions.

Lysophosphatidylcholine Inhibits Endothelial Cell Migration and Proliferation via Inhibition of the Extracellular Signal–Regulated Kinase Pathway

Lysophosphatidylcholine (lysoPC), a major lipid component of oxidized low density lipoprotein, inhibits endothelial cell (EC) migration and proliferation, which are critical processes during angiogenesis and the repair of injured vessels. However, the mechanism(s) of lysoPC-induced inhibition of EC migration and proliferation has not been clarified. In this report, we demonstrate the critical role of extracellular signal-regulated kinase (ERK) in growth factor-stimulated EC migration and proliferation as well as their inhibition by lysoPC. EC migration and proliferation stimulated by basic fibroblast growth factor (FGF-2) were blocked by inhibition of ERK activity by both the specific mitogen-activated protein kinase kinase (MEK) 1 inhibitor PD98059 and the overexpression of a dominant-negative mutant of MEK1. Conversely, overexpression of a constitutively active mutant of MEK1 increased EC migration and proliferation, which were comparable to those of ECs stimulated with FGF-2. LysoPC inhibited FGF-2-induced ERK activation via prevention of Ras activation without inhibiting tyrosine phosphorylation of phospholipase C-gamma. Taken together, our data demonstrate that ERK activity is required for FGF-2-induced EC migration and proliferation and suggest that inhibition of the Ras/ERK pathway by lysoPC contributes to the reduced EC migration and proliferation.

Overexpression of Endothelial Nitric Oxide Synthase in Endothelial Cells Is Protective against Ischemia- Reperfusion Injury in Mouse Skeletal Muscle

Microvascular injury has been proposed to be a main cause of ischemia-reperfusion (I/R) injury. The roles of endothelial nitric oxide synthase (eNOS)-derived NO, a key regulator of vascular function, in I/R injury are incompletely understood. We used transgenic mice overexpressing eNOS in endothelial cells (eNOS-Tg) and their littermates wild-type mice (WT) to investigate the roles of eNOS in I/R injury in skeletal muscle. Superoxide levels in the affected muscles were reduced by approximately 50% in eNOS-Tg compared with WT during reperfusion. In WT, the disassembly of endothelial junctional proteins seen in the early period of reperfusion was recovered in the later phase. These findings were correlated with the increased vascular permeability in vivo. In contrast, eNOS-Tg maintained the endothelial junction assembly as well as vascular permeability during reperfusion. Leukocyte extravasation into tissue and up-regulated expression of adhesion molecules in the reperfused vessels were significantly inhibited in eNOS-Tg. Tissue viability of the affected muscle was decreased in WT time-dependently after reperfusion, whereas eNOS-Tg showed no significant reduction. NOS inhibition completely reversed these protective effects of eNOS overexpression in I/R injury. Thus, eNOS overexpression appears to prevent the I/R injury in skeletal muscle by maintaining vascular integrity.

Serotonin-Induced Hypercontraction Through 5-Hydroxytryptamine 1B Receptors in Atherosclerotic Rabbit Coronary Arteries

Background — Augmented vasoconstriction to serotonin (5-hydroxytryptamine [5-HT]) in atherosclerotic vessels plays a crucial role in the development of myocardial ischemia. We investigated mechanisms for serotonin-evoked hypercontraction in atherosclerotic rabbit coronary arteries. Methods and Results — Contractile responses to serotonergic agents of endothelium-denuded coronary arteries from control and Watanabe heritable hyperlipidemic rabbits (WHHL) were examined. WHHL coronary arteries exhibited hypercontraction to 5-HT1–receptor agonists; the constrictor threshold concentrations and ED50 to serotonin, 5-carboxamidotryptamine, and sumatriptan in WHHL were significantly lower, and the Emax in WHHL to these agents were increased 55% to 59% above those of the control. Serotonin-evoked contractions in both groups were inhibited by GR127935 (5-HT1B/1D antagonist; 0.1 to 1 nmol/L) and pertussis toxin but not by ketanserin (5-HT2 antagonist; 0.01 to 1 &mgr;mol/L), suggesting that the hypercontraction is most likely mediated by 5-HT1B/1D receptors through a pertussis toxin–sensitive pathway. Furthermore, simultaneous measurements of [Ca2+]i and isometric tension of fura-2–loaded arteries revealed that the hypercontraction was concomitant with the augmented elevation of [Ca2+]i in the smooth muscle. The 5-HT1B mRNA levels in WHHL coronary arteries increased to 2.5-fold over those in control arteries, whereas neither 5-HT1D nor 5-HT2A mRNA was detected in either group. Conclusions — Atherosclerotic rabbit coronary arteries exhibited the enhancement in contraction and Ca2+ mobilization in response to serotonin. The 5-HT1B receptor, which is upregulated by atherosclerosis, most likely mediates the augmenting effects of serotonin.